Podophyllotoxin is a well-known lignan which has been isolated from plant extracts, particularly from so-called Podophyllum resins obtained by solvent extraction of various parts--notably the roots and rhizomes--of plants of the genus Podophyllum, e.g. the North American species Podophyllum peltatum and the Indian species Podophyllum emodi.
Podophyllotoxin has proved to be a highly effective chemotherapeutic agent for the treatment of venereal warts (condylomata acuminata; also known as genital warts) [see, e.g., R. K. Beutner and G. von Krog, Seminars in Dermatol. 9 (1990) 148]. Another important current use of podophyllotoxin is as a starting material for the synthesis of the anti-cancer drugs known as VP 16-213 ("etoposide") and VM 26 ("teniposide") [see, e.g., B. F. Issell, Cancer Chemother. Pharmacol. 7 (1982) 73].
The use of podophyllotoxin as a drug in its own right, and as a starting material for the preparation of other important drugs, has led to considerable interest in the preparation of podophyllotoxin in pure form, and the patent literature contains a number of references, e.g. U.S. Pat. No. 4,680,399 (to Buchardt) and U.S. Pat. No. 5,057,616 (to Jennings et al.), relating hereto.
Podophyllotoxin has been reported to occur in a variety of polymorphic forms having different melting points, and in the form of various solvates [see, e.g., A. W. Schrecker et al., J. Org. Chem. 21 (1956) 288]. Thus, Schrecker et al. (in the latter reference) recognized at least four crystalline modifications of podophyllotoxin, viz.: A, with water (m.p. 161.degree.-162.degree. C.); B, unsolvated (m.p. 183.degree.-184.degree. C.); C, with water and benzene of crystallization (m.p. 114.degree.-118.degree. C. "foaming"); and D, unsolvated (m.p. 188.degree.-189.degree. C.).
Podophyllotoxin has also been found to retain not only water alone, but also water together with certain organic solvents--notably simple aromatic and heteroaromatic molecules, e.g. benzene, toluene, nitrobenzene, chlorobenzene, phenol and pyridine--in the crystal structure as so-called complexes (also known as "host-guest complexes" or "inclusion complexes") or solvates see, e.g., U.S. Pat. No. 4,680,399 and K. V. Andersen et al., J. Chem. Soc. Perkin Trans. 2 (1990) 1871. The ratio between podophyllotoxin, organic solvent and water in such complexes is frequently 2:1:2; thus, for example and by way of illustration, the crystalline toluene complex (which has the latter composition) contains approximately 100,000 ppm of toluene.
Andersen et al. (in the latter reference) have demonstrated by X-ray diffraction that unsolvated podophyllotoxin (C.sub.22 H.sub.22 O.sub.8) has a melting point in the range of 182.degree.-184.degree. C., and that podophyllotoxin semihydrate (C.sub.22 H.sub.22 O.sub.8.0.5H.sub.2 O) melts in the range of 162.degree.-164.degree. C.
Furthermore, and of particular significance in relation to the present invention (vide infra), a number of organic solvents become tenaciously adsorbed to or occluded on the surface of crystals of podophyllotoxin, and this has been confirmed by the present inventors for, for example, halogenated lower alkanes such as dichloromethane and chloroform. Moreover, this tenacity is often such that simple recrystallisation of such podophyllotoxin products having adsorbed solvent molecules using other solvents, e.g. alcohols, generally does not lead to satisfactory removal of the adsorbed solvent. The present inventors have found that the mere fact that a particular product exhibits a melting point in the correct range for pure, crystalline, anhydrous, solvent-free podophyllotoxin is no guarantee for the absence of adsorbed or occluded solvent, since a number of podophyllotoxin products with significant contents of absorbed halocarbon solvent have been found to exhibit the correct melting point interval for completely pure podophyllotoxin.
Failure on the part of various past investigators to recognize the occurrence of these various forms, solvates and "solvent adsorbates" has resulted in considerable confusion, as well as misinterpretation of results. Furthermore, and rather significantly from a pharmaceutical viewpoint, important patent literature methods for the preparation of podophyllotoxin, such as those described in U.S. Pat. No. 4,680,399 and U.S. Pat. No. 5,057,616, disclose the use of, inter alia, halocarbon solvents at a late stage in the isolation procedure, there being no disclosure in these documents to indicate any awareness of the fact that (as mentioned above) such solvents are tanaciously retained by crystals of (otherwise pure) podophyllotoxin.
U.S. Pat. No. 5,057,616 discloses (see column 5, lines 62-65) that the allegedly pure product produced by precipitation from a halocarbon solvent (such as dichloromethane) and subsequent recrystallization from, typically, a ketone/ether mixture "may have a somewhat low melting point", necessitating the use of a rather elaborate and time-consuming further crystallization and vacuum-drying procedure to arrive at a product displaying the correct melting point. In U.S. Pat. No. 4,680,399, which initially exploits the crystallization properties of podophyllotoxin complexes with simple aromatic and heteroaromatic molecules (yide supra) in a first stage of isolation of podophyllotoxin from Podophyllum resin, podophyllotoxin of "especially high purity" is isolated by simple recrystallization (from a solvent such as ethyl acetate or aqueous ethanol) of a product obtained, for example, from a chloroform solution of such a complex.
What appears to be the first reported attempt to prepare pure, anhydrous podophyllotoxin was made by W. R. Dunstan and T. A. Henry [J. Chem. Soc. (1898) 209] by recrystallization of a podophyllotoxin hydrate from "absolute ethanol". However, the crystals obtained by allowing the solvent to evaporate in "a vacuous desiccator" melted at 157.degree. C., and recrystallization of these crystals from alcohol by adding water resulted in podophyllotoxin in hydrated form. These authors go on to describe the preparation of "anhydrous podophyllotoxin" by heating the "hydrated substance" at its melting point (117.degree. C.) for a few minutes, dissolving the resulting product in dry chloroform, and adding light petroleum until the mixture became slightly turbid. Upon standing, the "anhydrous podophyllotoxin" crystallized.
Schrecker et al. [J. Org. Chem. 21 (1956) 288] describe the preparation of "unsolvated" (unhydrated) podophyllotoxin by a procedure involving heating hydrated podophyllotoxin at 137.degree. C. in a vacuum. However, not only is this method impracticable for the large scale preparation of anhydrous podophyllotoxin, but based on present knowledge it is apparent that prolonged treatment of podophyllotoxin at the elevated temperature in question will, owing to the thermolability of podophyllotoxin at such temperatures, at least lead to significant epimerisation [see, e.g., O. Buchardt et al., J. Pharm. Sci. 75 (1986) 1076].
On the basis of the above discussion, it is clear that there is a need for a straightforward method for the preparation--from any readily available intermediate podophyllotoxin product, such as a hydrate, an inclusion complex with an organic solvent, or another solvent-containing podophyllotoxin phase--of anhydrous podophyllotoxin which is of high and reproducible purity, and which is free of pharmaceutically unacceptable organic solvents. The present invention fulfils this need.